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Metabolically engineered Escherichia coli for efficient production of glycosylated natural products.

Peirú S, Rodríguez E, Menzella HG, Carney JR, Gramajo H - Microb Biotechnol (2008)

Bottom Line: Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides.However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts.By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Division, IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.

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LC/MS/MS analysis of TDP‐sugars in cell‐free extracts of different E. coli strains: (A) K207‐3; (B) LB1b; (C) LB9b; (D) LB11b. m/z parent/daughter pairs for each compound are indicated.
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f2: LC/MS/MS analysis of TDP‐sugars in cell‐free extracts of different E. coli strains: (A) K207‐3; (B) LB1b; (C) LB9b; (D) LB11b. m/z parent/daughter pairs for each compound are indicated.

Mentions: K207‐3 is an E. coli B‐derived strain originally developed for the heterologous expression of polyketide biosynthetic genes, and further tested for the production of fully decorated polyketides (Murli et al., 2003; Peiru et al., 2005). However, the polyketide glycosylation efficiency of this strain is very low. To optimize this strain for increased production of novel TKDG‐derived TDP‐sugars, we first decided to increase the intracellular levels of TKDG by identifying and then disrupting the competing endogenous pathways. Although the genes involved in the biosynthesis of TDP‐l‐rhamnose and TDP‐d‐Qui4NAc have not been identified, previous works revealed the presence of these TDP‐sugars in E. coli B, indicating that their biosynthetic pathways would remain functional (Fig. 2A, see below; Okazaki et al., 1960). Moreover, even though E. coli strain B does not display O‐specific side‐chain LPS, genetic evidence suggests that this strain could be a former O7 E. coli, in which its O antigen synthesis was inactivated by an insertion sequence (Marolda et al., 1999; Schneider et al., 2002). Therefore, based on the available sequencing data from the O7‐specific LPS biosynthetic gene cluster of E. coli VW187 (Fig. 1B), we designed primers for the isolation of the rmlC and vioA genes, involved in the synthesis of TDP‐l‐rhamnose and TDP‐d‐Qui4NAc respectively. rmlC encodes the enzyme that catalyses 3′,5′‐epimerization of TKDG in the TDP‐l‐rhamnose pathway, while vioA encodes the TKDG aminotransferase involved in the synthesis of TDP‐d‐Qui4NAc (Fig. 1A) (Marolda et al., 1999; Wang et al., 2007). PCR products were obtained using K207‐3 chromosomal DNA and their nucleotide sequences were 100% identical to the previously reported rmlC and vioA gene sequences from the O7‐specific LPS biosynthesis gene cluster of E. coli VW187.


Metabolically engineered Escherichia coli for efficient production of glycosylated natural products.

Peirú S, Rodríguez E, Menzella HG, Carney JR, Gramajo H - Microb Biotechnol (2008)

LC/MS/MS analysis of TDP‐sugars in cell‐free extracts of different E. coli strains: (A) K207‐3; (B) LB1b; (C) LB9b; (D) LB11b. m/z parent/daughter pairs for each compound are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815289&req=5

f2: LC/MS/MS analysis of TDP‐sugars in cell‐free extracts of different E. coli strains: (A) K207‐3; (B) LB1b; (C) LB9b; (D) LB11b. m/z parent/daughter pairs for each compound are indicated.
Mentions: K207‐3 is an E. coli B‐derived strain originally developed for the heterologous expression of polyketide biosynthetic genes, and further tested for the production of fully decorated polyketides (Murli et al., 2003; Peiru et al., 2005). However, the polyketide glycosylation efficiency of this strain is very low. To optimize this strain for increased production of novel TKDG‐derived TDP‐sugars, we first decided to increase the intracellular levels of TKDG by identifying and then disrupting the competing endogenous pathways. Although the genes involved in the biosynthesis of TDP‐l‐rhamnose and TDP‐d‐Qui4NAc have not been identified, previous works revealed the presence of these TDP‐sugars in E. coli B, indicating that their biosynthetic pathways would remain functional (Fig. 2A, see below; Okazaki et al., 1960). Moreover, even though E. coli strain B does not display O‐specific side‐chain LPS, genetic evidence suggests that this strain could be a former O7 E. coli, in which its O antigen synthesis was inactivated by an insertion sequence (Marolda et al., 1999; Schneider et al., 2002). Therefore, based on the available sequencing data from the O7‐specific LPS biosynthetic gene cluster of E. coli VW187 (Fig. 1B), we designed primers for the isolation of the rmlC and vioA genes, involved in the synthesis of TDP‐l‐rhamnose and TDP‐d‐Qui4NAc respectively. rmlC encodes the enzyme that catalyses 3′,5′‐epimerization of TKDG in the TDP‐l‐rhamnose pathway, while vioA encodes the TKDG aminotransferase involved in the synthesis of TDP‐d‐Qui4NAc (Fig. 1A) (Marolda et al., 1999; Wang et al., 2007). PCR products were obtained using K207‐3 chromosomal DNA and their nucleotide sequences were 100% identical to the previously reported rmlC and vioA gene sequences from the O7‐specific LPS biosynthesis gene cluster of E. coli VW187.

Bottom Line: Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides.However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts.By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Division, IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.

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Related in: MedlinePlus